Team:Slovenia/Notebook

From 2012.igem.org

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<li>50 – 400 ng DNA solution was added to competent bacterial cells (depending on the concentration of the DNA solution). </li>
<li>50 – 400 ng DNA solution was added to competent bacterial cells (depending on the concentration of the DNA solution). </li>
<li>A mixture of cells and DNA solution was incubated on ice for 30-60 minutes. </li>
<li>A mixture of cells and DNA solution was incubated on ice for 30-60 minutes. </li>
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<li>The mixture was heat-shocked for 3 min at 42 °C. </li>
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<li>The mixture was heat-shocked for 3 minutes at 42 °C. </li>
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<li>Cooled for 3 minute on ice. </li>
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<li>Cooled for 3 minutes on ice. </li>
<li> 500 µL  of preheated antibiotic free LB-medium was added and incubated for one hour at 37 °C with agitation for the purpose of inducing antibiotic resistance. </li>
<li> 500 µL  of preheated antibiotic free LB-medium was added and incubated for one hour at 37 °C with agitation for the purpose of inducing antibiotic resistance. </li>
<li>The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates. </li>
<li>The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates. </li>
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<ol>
<ol>
<li>1 mL of an overnight culture was added to 150 µL of 80% glycerol into a cryo-tube. </li>
<li>1 mL of an overnight culture was added to 150 µL of 80% glycerol into a cryo-tube. </li>
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<li>Mixed and incubated at room temperature for 30 min. </li>
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<li>Mixed and incubated at room temperature for 30 minutes. </li>
<li>Afterwards the glycerol stock was stored at -80 °C. </li>
<li>Afterwards the glycerol stock was stored at -80 °C. </li>
</ol>
</ol>
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<h3>Eucaryotic cell lines and cultivation</h3>
<h3>Eucaryotic cell lines and cultivation</h3>
<p><b>HEK293</b> is a human cell line derived from kidney cells and grows in a monolayer culture. Cells were grown in DMEM medium supplemented with 10% FBS. </p>
<p><b>HEK293</b> is a human cell line derived from kidney cells and grows in a monolayer culture. Cells were grown in DMEM medium supplemented with 10% FBS. </p>
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<p><b>HEK293T</b> cell line is derived form HEK293 cells. HEK293T cells express the SV40 large T-antigen that enables episomal replication of plasmids containing the SV40 origin of replication in transfected cells. Cells were grown in DMEM medium supplemented with 10% FBS. </p>
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<p><b>HEK293T</b> cell line is derived from HEK293 cells. HEK293T cells express the SV40 large T-antigen that enables episomal replication of plasmids containing the SV40 origin of replication in transfected cells. Cells were grown in DMEM medium supplemented with 10% FBS. </p>
<p><b>NK-92</b> is an interleukin-2 (IL-2) dependent natural killer cell line derived from peripheral blood mononuclear cells from patient with non-Hodgkin's lymphoma. The cell line is cytotoxic to a wide range of malignant cells. Cells were grown in RPMI medium supplemented with 20% FBS and 100 U/ml IL-2.</p>
<p><b>NK-92</b> is an interleukin-2 (IL-2) dependent natural killer cell line derived from peripheral blood mononuclear cells from patient with non-Hodgkin's lymphoma. The cell line is cytotoxic to a wide range of malignant cells. Cells were grown in RPMI medium supplemented with 20% FBS and 100 U/ml IL-2.</p>
</br>
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<li>Remove and discard culture medium from a T-75 flask containing a monolayer of HEK293 or HEK293T cells. </li>
<li>Remove and discard culture medium from a T-75 flask containing a monolayer of HEK293 or HEK293T cells. </li>
<li>Rinse the T-75 flask with 10 ml of PBS buffer to remove all traces of growth medium (DMEM + 10% FBS) which otherwise inhibits trypsin function. Remove and discard the PBS buffer. </li>
<li>Rinse the T-75 flask with 10 ml of PBS buffer to remove all traces of growth medium (DMEM + 10% FBS) which otherwise inhibits trypsin function. Remove and discard the PBS buffer. </li>
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<li>Add 2-3 ml of trypsine solution and gently tilt the flask to ensure the trypsine solution covers all the cells. Incubate the cells in trypsin for 0,5 - 3 min. </li>
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<li>Add 2-3 ml of trypsine solution and gently tilt the flask to ensure the trypsine solution covers all the cells. Incubate the cells in trypsin for 0,5 - 3 minutes. </li>
<li>When the cells start to detach from the surface, add 7 ml of growth medium to the trypsin solution. Resuspend all remaining cells from the bottom of the T-75 flask by pipetting. </li>
<li>When the cells start to detach from the surface, add 7 ml of growth medium to the trypsin solution. Resuspend all remaining cells from the bottom of the T-75 flask by pipetting. </li>
<li>Transfer the cell suspension to a 15 ml centrifuge tube. </li>
<li>Transfer the cell suspension to a 15 ml centrifuge tube. </li>
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<li>Centrifuge the cell suspension for 5 min at 1200 rpm. </li>
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<li>Centrifuge the cell suspension for 5 minutes at 1200 rpm. </li>
<li>Remove the trypsin-containing medium from the centrifuge tube. </li>
<li>Remove the trypsin-containing medium from the centrifuge tube. </li>
<li>Resuspend the cell pellet in fresh medium. </li>
<li>Resuspend the cell pellet in fresh medium. </li>
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<h3>Induction systems</h3>
<h3>Induction systems</h3>
   
   
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<p>To control our switch we needed a way to affect it from the outside. For this purpose we chose several inducible transcription systems where the controlled gene is expressed when a small molecule inducer (such as tetracycline) is present and is not expressed when the inducer is absent. We chose specific systems which do not cross react and whose inducers are orally bioavailable and safe for human use (Clackson, 2000). The systems we chose are based on tetracycline, pristinamycin, erythromycin and rapamycin analogs, as inducers. We then adapted these systems by cloning TAL regulators under their control to make them compatible with our genetic circuits. </p>
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<p>To control our switch we needed a way to affect it from the outside. For this purpose we chose several inducible transcription systems where the controlled gene is expressed when a small molecule inducer (such as tetracycline) is present and is not expressed when the inducer is absent. We chose specific systems which do not cross react and whose inducers are orally bioavailable and safe for human use (Clackson, 2000). We decided for the systems based on tetracycline, pristinamycin, erythromycin and rapamycin analogs, as inducers. We then adapted these systems by cloning TAL regulators under their control to make them compatible with our genetic circuits. </p>
   
   
<h3>Tetracycline, erythromycin and pristinamycin systems</h3>
<h3>Tetracycline, erythromycin and pristinamycin systems</h3>
   
   
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<p>The tetracycline, erythromycin and pristinamycin system all function in a similar manner. They are composed of a DNA binding protein (such as TetR) fused to a KRAB domain which reversibly binds a specific DNA sequence (TRE for example) and silences transcription from nearby promoters. The addition of an inducer causes the DNA binding domain to dissociate from the DNA and allows transcription to start. (Deuschle et al., 1995) (Kramer et al., 2004) </p>
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<p>The tetracycline, erythromycin and pristinamycin system all function in a similar manner. They are composed of a DNA binding protein (such as TetR) fused to a KRAB domain which reversibly binds a specific DNA sequence (TRE for example) and silences transcription from nearby promoters. The addition of an inducer causes the DNA binding domain to dissociate from the DNA and allows transcription to start. (Deuschle et al., 1995; Kramer et al., 2004) </p>
   
   
   
   
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<h3>Rapamycin system</h3>
<h3>Rapamycin system</h3>
   
   
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<p>In the rapamycin system the gene of interest is under the control of a minimal promoter. The gene's transcription rate is regulated by two proteins that consist of a drug binding domain and either a DNA binding domain or an activation domain. When rapamycin is added both drug binding domains bind to it, consequently joining the activation domain with the DNA binding domain, resulting in a functional transcription factor, which activates the gene of interest. Instead of rapamycin a rapamycin analogue (rapalogue), which is a 1000-fold less imunosupressive than rapamycin, but activates the inducible system like rapamycin, is usualy used as the inducer. (Pollock et al., 2002) </p>
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<p>In the rapamycin system the gene of interest is under the control of a minimal promoter. The gene's transcription rate is regulated by two proteins that consist of a drug binding domain and either a DNA binding domain or an activation domain. When rapamycin is added both drug binding domains bind to it, consequently joining the activation domain with the DNA binding domain, resulting in a functional transcription factor, which activates the gene of interest. Instead of rapamycin a rapamycin analogue (rapalogue), which is a 1000-fold less imunosupressive than rapamycin, but activates the inducible system like rapamycin, is usually used as the inducer. (Pollock et al., 2002) </p>
   
   
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<ol>
<ol>
<li>Transfect HEK293 or HEK293T cells with plasmids using JetPei transfection reagent (Polyplus transfection), following the manufacturers protocol (see cell culturing for details). </li>
<li>Transfect HEK293 or HEK293T cells with plasmids using JetPei transfection reagent (Polyplus transfection), following the manufacturers protocol (see cell culturing for details). </li>
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<li>2 hours post transfection change media and stimulate the cells by adding dilutions of appropriate inductors to the medium in a 1:10 (v:v). </li>
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<li>Two hours post transfection change media and stimulate the cells by adding dilutions of appropriate inductors to the medium in a 1:10 (v:v). </li>
</ol>
</ol>
</br>
</br>
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<li>Additionally, we performed a co-transfection experiment, where HEK293T cells were transfected with both the reporter and the IFN-alpha encoding plasmids. </li>
<li>Additionally, we performed a co-transfection experiment, where HEK293T cells were transfected with both the reporter and the IFN-alpha encoding plasmids. </li>
<li>Day after transcfection cells were cultivated into 96-well plate at density 5x104 cells per well.</li>
<li>Day after transcfection cells were cultivated into 96-well plate at density 5x104 cells per well.</li>
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<li>After 24 hours of incubation, dual luciferase reporter assay was preformed. </li>
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<li>After 24 hours of incubation, dual luciferase reporter assay was performed. </li>
</ol>
</ol>
</br>
</br>
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<li>After a maximum of 6 days after transfection, cells were lysed with 25 µL of 1x Passive lysis buffer (Promega). </li>
<li>After a maximum of 6 days after transfection, cells were lysed with 25 µL of 1x Passive lysis buffer (Promega). </li>
<li>Luminescence of expressed reporter firefly luciferase was measured with Orion (Berthold Technologies) using Luciferase buffer with luciferin as a substrate. For normalization Renilla luciferase activity was used. The Renilla luciferase was measured using Renilla buffer supplemented with coelenterazine. </li>
<li>Luminescence of expressed reporter firefly luciferase was measured with Orion (Berthold Technologies) using Luciferase buffer with luciferin as a substrate. For normalization Renilla luciferase activity was used. The Renilla luciferase was measured using Renilla buffer supplemented with coelenterazine. </li>
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</ol>
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</br>
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<h3>Plate reader-absorbance  (The Switch) </h3>
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<ol>
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<li>HEK293T cells were seeded in white 24-well plates and transfected with plasmids encoding the positive feedback loop switch and 10x[TALB + TALC] operator_CMV promoter_fLuciferase reporter plasmid and 10x[TALA + TALC]operator_CMV promoter_SEAP plasmid. Plasmids and amounts used for transfection are listed in Figure legends.</li>
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<li>Media supplemented with inducer or without inducer were changed after two days of cultivation.</li>
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<li>Two and seven days after transfection the growth medium was collected and SEAP QUANTIBlue substrate was added. After 15 minutes incubation at 37°C  the absorbance was measured at 630 nm. </li>
</ol>
</ol>
</br>
</br>

Revision as of 16:41, 26 October 2012